PROJECT SUMMARY A highly effective malaria vaccine is likely required to eradicate the disease. Transmission blocking vaccines (TBVs) induce antibodies that block parasite reproduction and development in the mosquito midgut and are part of the World Health Organization Malaria Vaccine Technology roadmap. This R01 response to program announcement PA-19-077: Accelerating Malaria Vaccine Discovery, which specifically encourages studies that will lead to discovery of new vaccine candidates that interrupt malaria transmission. Pfs48/45 is a TBV candidate antigen, and is the target of one of the most potent transmission-blocking monoclonal antibodies (85RF45.1). Pfs48/45 has not yet been tested in humans, in part due to difficulties in producing the antigen. We will follow-up on our recent advances in producing first-in-class, well-defined, fusion-free Pfs48/45. Serendipitously, glycosylation at a non-essential epitope of the protein led to drastically enhanced protein yield, enabling us to solve its three-dimensional structure, also revealing the target epitope of 85RF45.1. This new- found structural information will be used to guide advanced antigen design, including stabilizing mutations and antigen design that is immunofocused to the potent neutralizing epitope. Rationally-optimized Pfs48/45 will be optimized in the context of a next-generation vaccine adjuvant. This adjuvant enables combining (via simple mixing) well-characterized antigens with a liposomal adjuvant to induce spontaneous nanoliposome-antigen particleization (SNAP). We discovered that liposomes that contain small amounts of cobalt porphyrin- phospholipid (CoPoP) bind to well-characterized his-tagged antigens with simple mixing via spontaneous insertion of the his-tag into the bilayer. Unlike other approaches, CoPoP liposomes give rise to rapid antigen particleization that is stable in biological media. His-tagged Pfs48/45 is simply mixed with CoPoP liposomes without further purification to convert the soluble antigen into a 100 nm particle with uniform, arrayed antigen display. In some immunization conditions, SNAP immunization induces orders of magnitude higher functional IgG responses with TBV antigens compared to other vaccine adjuvants. Antibody durability compares favorably to current toxin conjugation strategies which have progressed into clinical studies. Using immuno- focused and stabilized Pfs48/45 constructs, particleization parameters will be assessed and iterative antigen improvement will be carried out. Another relevant attribute of the SNAP approach that will be investigated is seamless multiplexing by simple mixing of multiple antigens at the time of immunization (without further purification), resulting in a balanced and functional IgG response (i.e. to each included antigen). The impact of multiplexing other TBV antigens (Pfs25 or Pfs230) on the Pfs48/45 response will be assessed, as will whether additive or synergistic functional activity is induced. Finally, the safety of SNAP immunization with the optimized Pfs48/45 construct and formulation will be tested.